Rosaria De Santis

Immunoglobulin superfamily receptors in protochordates: before RAG time.

Summary:  Urochordates and cephalochordates do not have an adaptive immune system involving the somatic rearrangement of their antigen receptor genes. They do not have antigen‐presenting molecules of the major histocompatibility complex (MHC)‐linked class I and II types. In the absence of such a system, the status of their genes reflects perhaps a primitive pre‐recombination‐activating gene (RAG) stage that could suggest the pathway leading to the genesis of the T‐cell receptor (TCR) and antibodies.

A study of the chorion and the follicle cells in relation to the sperm-egg interaction in the ascidian, Ciona intestinalis☆

Abstract We present ultrastructural observations on the processes underlying interaction between the spermatozoon and the egg envelopes in Ciona intestinalis. The morphological and cytochemical basis of sperm binding to the chorion and the subsequent steps such as the acrosome reaction and penetration of the spermatozoon through the chorion are described. Some speculations are also presented on the role of the follicle cells in fertilization.

Complement in urochordates: cloning and characterization of two C3-like genes in the ascidian Ciona intestinalis

The recent identification of complement components in deuterostome invertebrates has indicated the presence of a complement system operating via an alternative pathway in echinoderms and tunicates and via a MBL-mediated pathway thus far identified only in tunicates. Here, we report the isolation of two C3-like genes, CiC3-1 and CiC3-2, from blood cell total RNA of the ascidian Ciona intestinalis. The deduced amino acid sequences of both Ciona C3-like proteins exhibit a canonical processing site for α and β chains, a thioester site with an associated catalytic histidine and a convertase cleavage site, thus showing an overall similarity to the other C3 molecules already characterized. Southern blotting analysis indicated that each gene is present as a single copy per haploid genome. In situ hybridization experiments showed that both CiC3-1 and CiC3-2 are expressed in one type of blood cell, the compartment cells. Two polyclonal antibodies, raised against two deduced peptide sequences in the α chain of CiC3-1 and CiC3-2, allowed the identification by Western blot of a single band in the blood serum, of about Mr 150,000. A phylogenetic tree, based on the alignment of CiC3-1 and CiC3-2 with molecules of the α2-macroglobulin superfamily, indicated that the Ciona C3s form a cluster with Halocynthia roretzi C3. The phylogenetic analysis also suggested that the duplication event from which the CiC3-1 and CiC3-2 genes originated occurred in the urochordate lineage after the separation of the Halocynthia and Ciona ancestor.

Store-operated calcium channel regulates the chemotactic behavior of ascidian sperm

The sperm-activating and -attracting factor released from the eggs of the ascidians Ciona intestinalis and Ciona savignyi requires extracellular Ca2+ for activating sperm motility and eliciting chemotactic behavior of the activated sperm toward the egg. Here, we show that modulators of the store-operated Ca2+ channel, SK&F96365, Ni2+, 2-aminoethoxydiphenylborane, and thapsigargin inhibit the chemotactic behavior of the ascidian sperm; on the other hand, blockers of voltage-dependent Ca2+ channels did not inhibit the chemotaxis, even though they inhibited the sperm activation operated by voltage-dependent Ca2+ channels. The blockers of store-operated Ca2+ channels also inhibited the asymmetrical flagellar beating and turning movements of the ascidian sperm, which are typical signs of sperm chemotaxis. Depletion of internal Ca2+ stores by thapsigargin induced capacitative Ca2+ entry into the sperm, which was blocked by SK&F96365. These results suggest that the intracellular Ca2+ concentration increase through the store-operated Ca2+ channels induces asymmetrical flagellar movements to establish the chemotactic behavior of the sperm.

CiC3-1a-Mediated Chemotaxis in the Deuterostome Invertebrate Ciona intestinalis (Urochordata)

Deuterostome invertebrates possess complement genes, and in limited instances complement-mediated functions have been reported in these organisms. However, the organization of the complement pathway(s), as well as the functions exerted by the cloned gene products, are largely unknown. To address the issue of the presence of an inflammatory pathway in ascidians, we expressed in Escherichia coli the fragment of Ciona intestinalis C3-1 corresponding to mammalian complement C3a (rCiC3-1a) and assessed its chemotactic activity on C. intestinalis hemocytes. We found that the migration of C. intestinalis hemocytes toward rCiC3-1a was dose dependent, peaking at 500 nM, and was specific for CiC3-1a, being inhibited by an anti-rCiC3-1a-specific Ab. As is true for mammalian C3a, the chemotactic activity of C. intestinalis C3-1a was localized to the C terminus, because a peptide representing the 18 C-terminal amino acids (CiC3-1a59–76) also promoted hemocyte chemotaxis. Furthermore, the CiC3-1a terminal Arg was not crucial for chemotactic activity, because the desArg peptide (CiC3-1a59–75) retained most of the directional hemocyte migration activity. The CiC3-1a-mediated chemotaxis was inhibited by pretreatment of cells with pertussis toxin, suggesting that the receptor molecule mediating the chemotactic effect is Gi protein coupled. Immunohistochemical analysis with anti-rCiC3-1a-specific Ab and in situ hybridization experiments with a riboprobe corresponding to the 3′-terminal sequence of CiC3-1, performed on tunic sections of LPS-injected animals, showed that a majority of the infiltrating labeled hemocytes were granular amebocytes and compartment cells. Our findings indicate that CiC3-1a mediates chemotaxis of C. intestinalis hemocytes, thus suggesting an important role for this molecule in inflammatory processes.

A role for variable region-containing chitin-binding proteins (VCBPs) in host gut-bacteria interactions.

A number of different classes of molecules function as structural matrices for effecting innate and adaptive immunity. The most extensively characterized mediators of adaptive immunity are the immunoglobulins and T-cell antigen receptors found in jawed vertebrates. In both classes of molecules, unique receptor specificity is effected through somatic variation in the variable (V) structural domain. V region-containing chitin-binding proteins (VCBPs) consist of two tandem Ig V domains as well as a chitin-binding domain. VCBPs are encoded at four loci (i.e., VCBPA–VCBPD) in Ciona, a urochordate, and are expressed by distinct epithelial cells of the stomach and intestine, as well as by granular amoebocytes present in the lamina propria of the gut and in circulating blood. VCBPs are secreted into the gut lumen, and direct binding to bacterial surfaces can be detected by immunogold analysis. Affinity-purified native and recombinant VCBP-C, as well as a construct consisting only of the tandem V domains, enhance bacterial phagocytosis by granular amoebocytes in vitro. Various aspects of VCBP expression and function suggest an early origin for the key elements that are central to the dialogue between the immune system of the host and gut microflora.

The gut of geographically disparate Ciona intestinalis harbors a core microbiota.

It is now widely understood that all animals engage in complex interactions with bacteria (or microbes) throughout their various life stages. This ancient exchange can involve cooperation and has resulted in a wide range of evolved host-microbial interdependencies, including those observed in the gut. Ciona intestinalis, a filter-feeding basal chordate and classic developmental model that can be experimentally manipulated, is being employed to help define these relationships. Ciona larvae are first exposed internally to microbes upon the initiation of feeding in metamorphosed individuals; however, whether or not these microbes subsequently colonize the gut and whether or not Ciona forms relationships with specific bacteria in the gut remains unknown. In this report, we show that the Ciona gut not only is colonized by a complex community of bacteria, but also that samples from three geographically isolated populations reveal striking similarity in abundant operational taxonomic units (OTUs) consistent with the selection of a core community by the gut ecosystem.

Studies on fertilization in the ascidians: Fucosyl sites on vitelline coat of Ciona intestinalis

Abstract The vitelline coat (originally called chorion) of the ascidian egg is the site where species-specific recognition and binding of spermatozoa occurs. Recent findings from this laboratory have suggested that fucosyl residues are present on the vitelline coat of Ciona intestinalis eggs and play an important role in the process of fertilization. The results reported in this paper confirm and extend those findings. With Fucose Binding Protein (FBP) and Fucosyl-Ferritin as markers, fucosyl sites have been localized on the fibrillar tufts emerging from the outer surface of the vitelline coat, both on glycerol-treated eggs and on living eggs deprived of follicle cells at pH 5. Observations on isolated vitelline coats have shown that the inner surface does not contain fucosyl sites. Studies performed with 125I-FBP on glycerol-treated eggs have indicated that two distinct classes of fucosyl sites are present on the vitelline coat. The association constants for FBP of the high affinity and of the low affinity class are 2.3×106 and 4.1×105 respectively. The gel electrophoresis of the proteins extracted in sodium dodecyl sulfate (SDS) from sonicated vitelline coats has shown three fucosyl-containing polypeptide bands.

Differentiation of the vitelline coat in the ascidianCiona intestinalis: an ultrastructural study

SummaryWe have studied the differentiation of the vitelline coat (VC) of the ascidianCiona intestinalis. In the young previtellogenic oocyte the vitelline coat precursor material (VCPM) makes its first appearance as patches of fibrous material in close apposition to the outer surface of the oocyte. The presence of subcortical vescicles containing a fuzzy electron-dense material and their opening into the oocyte surface parallels the formation of VCPM. Numerous microvillar-like structures emerge from the oocyte surface. When the VCPM completely surrounds the oocyte the microvilli are withdrawn. An overall increase of VCPM parallels the growth of the oocyte. The next step in the differentiation of the vitelline coat consists in the packing of the constituent fibrils in a dense layer at its outer surface, i.e. the one in contact with the follicle cells. At this time the VC is penetrated by microvilli protruding both from the oocyte and follicle cells. The VC reaches its final structure and thickness at the time the test cells are extruded into the perivitelline space.The participation of the follicle cells in VC organization is also discussed.

First Identification of a Chemotactic Receptor in an Invertebrate Species: Structural and Functional Characterization of Ciona intestinalis C3a Receptor

In mammals, the bioactive fragment C3a, released from C3 during complement activation, is a potent mediator of inflammatory reactions and exerts its functional activity through the specific binding to cell surface G protein-coupled seven-transmembrane receptors. Recently, we demonstrated a Ciona intestinalis C3a (CiC3a)-mediated chemotaxis of hemocytes in the deuterostome invertebrate Ciona intestinalis and suggested an important role for this molecule in inflammatory processes. In the present work, we have cloned and characterized the receptor molecule involved in the CiC3a-mediated chemotaxis and studied its expression profile. The sequence, encoding a 95,394 Da seven-transmembrane domain protein, shows the highest sequence homology with mammalian C3aRs. Northern blot analysis revealed that the CiC3aR is expressed abundantly in the heart and neural complex and to a lesser extent in the ovaries, hemocytes, and larvae. Three polyclonal Abs raised in rabbits against peptides corresponding to CiC3aR regions of the first and second extracellular loop and of the third intracellular loop react specifically in Western blotting with a single band of 98–102 kDa in hemocyte protein extracts. Immunostaining performed on circulating hemocytes with the three specific Abs revealed that CiC3aR is constitutively expressed only in hyaline and granular amoebocytes. In chemotaxis experiments, the Abs against the first and second extracellular loop inhibited directional migration of hemocytes toward the synthetic peptide reproducing the CiC3a C-terminal sequence, thus providing the compelling evidence that C. intestinalis expresses a functional C3aR homologous to the mammalian receptor. These findings further elucidate the evolutionary origin of the vertebrate complement-mediated proinflammatory process.